Effects of two auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) and α-napthaleneacetic acid (NAA) on nicotine production during callus culture of a wild tobacco (Nicotiana glauca) were investigated using a high performance liquid chromatography (HPLC). The high concentration (11.5 μM) of 2,4-D and NAA had peaks of nicotine contents at 4th and 2nd week, respectively. Thereafter, the concents decreased and the nicotine was metabolized to other alkaloids, The low concentration (1.5 μM) of 2,4-D on the medium supplemented with 0.1 mM of L-aspartic acid of L-arginine inhibited nicotine production. However, the low NAA promoted it only when the medium was supplemented with L-aspartic acid. From these results, it could be concluded that both auxins exhibit different action mechanisms on nicotine production pathway and the low NAA promotes the activities for the pathway with L-aspartic acid as a precursor.